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The nucleotide phosphodiesterase/pyrophosphatase from Xanthomonas axonopodis (NPP) is a structural and evolutionary relative of alkaline phosphatase that preferentially hydrolyzes phosphate diesters. With the goal of understanding how these two enzymes with nearly identical Zn[sup2+] bimetallo sites achieve high selectivity for hydrolysis of either phosphate monoesters or diesters, we have measured a promiscuous sulfatase activity in NPP. Sulfate esters are nearly isosteric with phosphate esters but carry less charge, offering a probe of electrostatic contributions to selectivity. NPP exhibits sulfatase activity with k[subcat]/K[subM] value of 2 x 10[sup-5] M[sup-1] s[sup-1], similar to the R166S mutant of alkaline phosphatase. We further report the effects of thio-substitution on phosphate monoester and diester reactions. Reactivities with these noncognate substrates illustrate a reduced dependence of NPP reactivity on the charge of the nonbridging oxygen situated between the Zn[sup2+] ions relative to that in alkaline phosphatase. This reduced charge dependence can explain about 10[sup2] of the 10[sup7]-fold differential catalytic proficiency for the most similar monoester and diester substrates in the two enzymes. The results further suggest that active site contacts to substrate oxygen atoms that do not contact the Zn[sup2+] ions may play an important role in defining the selectivity of the enzymes. [ABSTRACT FROM AUTHOR]

The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline phosphatase (AP) using linear free-energy relationships (LFERs). We determined kcat/KM for a series of awl sulfate ester monoanions to obtain the Brønsted coefficient, βig, and compared the value to that obtained previously for a series of awl phosphorothioate ester dianion substrates. Despite the difference in substrate charge, the observed Brønsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis (-0.76 ± 0.14 and -0.77 ± 0.10, respectively) are strikingly similar, with steric effects being responsible for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition states in solution. These observations suggest an apparent equivalency of the transition states for phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active site electrostatic interactions on charge distribution in the transition state. [ABSTRACT FROM AUTHOR]

This article reports that alkaline phosphatase (AP) catalysis is ultrasensitive to charge sequestered between the active site zinc ions. Escherichia coli AP employs a binuclear metallocenter of two zinc ions to facilitate catalysis of phosphate monoester hydrolysis. Indeed, AP has been found to catalyze reactions beyond phosphate monoester hydrolysis. Earlier studies of catalytic promiscuity and the AP catalytic mechanism suggested that substrates with reduced charge on the non-bridging oxygen atoms were less efficiently hydrolyzed relative to dianionic phosphate monoesters. Although it is tempting to describe increased electrostatic sensitivity, such as that observed in the AP active site, in terms of a reduced dielectric constant relative to water, the bulk concept of a dielectric is not applicable to idiosyncratic environments of atomic scale such as enzyme active sites.